Endogenous TGF-beta activity is modified during cellular aging: effects on metalloproteinase and TIMP-1 expression

In culture, nontransformed human diploid fibroblasts divide a limited number of times, resulting in a nonproliferating senescent cell culture which exhibits an altered pattern of gene expression. Previously we reported that an early event in the process of replicative senescence was an increase in the synthesis of two connective tissue degrading metalloproteinases, collagenase and stromelysin, and a decrease in the synthesis of the physiological inhibitor of those enzymes, tissue inhibitor of metalloproteinases-1 (TIMP-1). The cytokine TGF-beta1 is known to regulate the expression of each of these three genes and to be synthesized and secreted by cultured human fibroblasts. This suggested the hypothesis that the age-specific modulation of collagenase, stromelysin, and TIMP-1 expression is the result of a change in TGF-beta1 activity during replicative senescence. To test this hypothesis, the responses of early, mid, and late passage (presenescent) fibroblast cell cultures to a TGF-beta neutralizing antibody were evaluated. In early passage cell cultures, exposure to TGF-beta neutralizing antibody resulted in a significant increase in the expression of collagenase and stromelysin and decreased TIMP-1 expression. The antibody did not affect expression of either of those genes by late passage cell cultures, although late passage cultures did respond to added TGF-beta1. Quantification of the levels of active TGF-beta, using a growth inhibition assay, indicates that the level of active TGF-beta1 is decreased during replicative senescence, supporting the conclusion that the modulation of collagenase, stromelysin, and TIMP-1 expression results from diminished TGF-beta activity.